The detection of target nucleic acid in test samples is important in various fields, including medicine and biology. Many compositions and procedures are available for the detection of specific nucleic acid molecules. Typically, this technology is based on sequence-dependent hybridization between a target nucleic acid and a nucleic acid probe which may range in length from short oligonucleotides (20 bases or less) to many kilobases (kb).
One widely used method for amplification of specific sequences from within a population of nucleic acid sequences is the polymerase chain reaction (PCR). In a typical PCR reaction, a target nucleic acid is amplified in three distinct steps: dissociation (denaturation) of a double-stranded template DNA into single strands; annealing of primers to the single-stranded template DNAs; and synthesis (extension) of a complementary strand from each primer. During PCR, the denaturation process, the annealing process, and the extension process are each carried out at different temperatures and are repeatedly cycled through the different reaction temperatures with a thermal cycler. Consequently, expensive temperature cycle control equipment is necessary to perform such a reaction, which prevents its routine use in field examinations, point-of-care (bedside) diagnoses, and inexpensive examinations.
Moreover, since the PCR reaction is carried out at three different temperatures, the reaction can be associated with challenges such as difficulty in maintaining accurate temperatures and that the time loss increases in proportion to the number of cycles. Further, the denaturation of a double-stranded template DNA into single strands requires the use of high temperatures, which necessitates that the reaction be performed using a limited number of thermostable DNA polymerases.
Accordingly, the following disclosure provides alternative methods and compositions for detecting a nucleic acid (such as DNA or RNA) under reaction conditions that are less rigorous than those used in PCR, while maintaining selectivity and sensitivity adequate to allow for the detection of nucleic acid molecules having a short length and at low concentrations. Among other aspects, the present disclosure provides novel methods and nucleic acid molecules that can improve the detection limit of target nucleic acids in a sample under low temperature, isothermal conditions, and can simplify or improve sample preparation and automated methods of detection.